Original Article-Metabolism
www.cmj.ac.kr
Effects of Epigallocatechin-3-Gallate on the Expression of TGF- β 1, PKC α / β II, and NF- κ B in High-Glucose-Stimulated Glomerular Epithelial Cells
Sung Jun Park, Ji Min Jeong2 , Han-Seong Jeong1, Jong-Seong Park1 and Nam-Ho Kim2*
School of Mechanical Systems Engineering, Chonnam National University, Departments of 1Physiology and 2Medicine, Chonnam National University Medical School, Gwangju, Korea
Epigallocatechin-3-gallate (EGCG) is the most potent antioxidant polyphenol in green tea. In the present study, we investigated whether EGCG plays a role in the expression of transforming growth factor-beta1 (TGF-β1), protein kinase C (PKC) α/βII, and nu- clear factor-kappaB (NF-κB) in glomerular epithelial cells (GECs) against high-glucose injury. Treatment with high glucose (30 mM) increased reactive oxygen species (ROS)/
lipid peroxidation (LPO) and decreased glutathione (GSH) in GECs. Pretreatment with 100 μM EGCG attenuated the increase in ROS/LPO and restored the levels of GSH, whereas ROS, LPO, and GSH levels were not affected by treatment with 30 mM man- nitol as an osmotic control. Interestingly, high-glucose treatment affected 3 separate signal transduction pathways in GECs. It increased the expression of TGF-β1, PKC α/βII, and NF-κB in GECs, respectively. EGCG (1, 10, 100 μM) pretreatment sig- nificantly decreased the expression of TGF-β1 induced by high glucose in a dose-de- pendent manner. In addition, EGCG (100 μM) inhibited the phosphorylation of PKC α/βII caused by glucose at 30 mM. Moreover, EGCG (1, 10, 100 μM) pretreatment sig- nificantly decreased the transcriptional activity of NF-κB induced by high glucose in a dose-dependent manner. These data suggest that EGCG could be a useful factor in modulating the injury to GECs caused by high glucose.
Key Words: Glomerular epithelial cell; Epigallocatechin-3-gallate; Transforming growth factor-β1; Protein kinase C α/βII; Nuclear factor-κB
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Article History:
received 17 May, 2011 accepted 14 July, 2011
Corresponding Author:
Nam-Ho Kim
Department of Medicine, Chonnam National University Medical School, 8 Hak 1-dong, Dong-gu, Gwangju 501-749, Korea
TEL: +82-62-220-6292 FAX: +82-62-225-8578 E-mail: [email protected]
INTRODUCTION
Glomerular epithelial cells (GECs) are highly speci- alized cells characterized by actin-rich foot processes that reside on the glomerular basement membrane. These cells have an important role in the maintenance of the structure and function of the glomerular filter.1 Injury to the glomer- ulus is characterized by the disappearance or effacement of the foot processes, leading to leakage of protein into the urine. Several inflammatory diseases, such as glomer- ulonephritis, and diabetic nephropathy result in GEC loss and glomerulosclerosis.2 Recent data suggest that the dia- betic milieu involves damage to GECs.3
Oxidative stress arises from the strong cellular oxidizing potential of excess reactive oxygen species (ROS) or free
radicals. Despite their toxic effects, ROS play important roles in signal transduction pathways, such as apoptosis, cell proliferation, and differentiation, and as defenders against bacterial invasion.4 Moreover, ROS induced by is- chemia-reperfusion or inflammation cause serious dam- age to tissues, including the kidneys.5 ROS contribute to stimulated cell death by damaging DNA and by stimulat- ing the lipoperoxidation of cellular membranes.6 Organi- sms respond to ROS through adaptation reactions, such as the stimulation of antioxidant proteins and antioxidant en- zymes, including reduced glutathione (GSH), superoxide dismutase (SOD), and catalase.7 However, the ROS-regu- lated signaling pathways leading to renal cellular re- sponses in the diabetic kidney are not entirely clear.
Transforming growth factor (TGF)-β is a multifunctional
signaling cytokine that may alter cell behavior by control- ling the growth, differentiation, death, and function of cells.8 Protein kinase C (PKC) is a family of serine/threo- nine kinases that are involved in a variety of pathways that regulate cell growth, death, and stress responsiveness.
Current evidence demonstrates that PKC is a sensitive tar- get for redox modification.9 Nuclear factor-κB (NF-κB) is a heterodimer that is activated in response to primary or secondary pathogenic stimuli.10
Epigallocatechin-3-gallate (EGCG) is the most abundant antioxidant polyphenol in green tea. It is a useful agent for protecting against protein oxidation-associated diseases.11 It has been shown to improve age-related cognitive decline, cerebral ischemia/reperfusion injuries,12 and diabetic nephropathy induced by streptozotocin (STZ) injection.13 Although the activity of EGCG in biological events has been investigated, its effect on signal transduction is not yet fully defined.
In the present study, we studied the close relation be- tween the antioxidant property of EGCG and the pro- tection of GECs exposed to high levels of glucose and tried to determine the possible intracellular signaling pathways implicated.
MATERIALS AND METHODS 1. Reagents
EGCG, D-glucose, mannitol, and dihydrodichlorofluore- scein diacetate (DCF-DA) were purchased from Sigma (St.
Louis, MO). Anti-phospho-PKC α/βII (Thr638/641) anti- body was purchased from Cell Signaling Technologies Inc.
(Beverly, MA), and anti-TGF-β1 antibody, anti-PKC-α an- tibody, and anti-GAPDH antibody were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA).
2. Cell culture
Rat glomerular visceral epithelial cells were isolated and characterized as previously described.14 Cells were used between the 10th and 20th passages and were cultured in Dulbecco’s modified Eagle’s medium (Life Technologies, Grand Island, NY) containing 10% fetal bovine serum (Cambrex Co., Walkersville, MD), 0.5 mg/ml Fungizone, and 100 U/ml penicillin, as described previously.15,16 When the cells reached 70% confluence in 100-mm culture dishes, they were incubated in serum-free media for 2 days to ar- rest and synchronize cell growth.
3. Measurement of intracellular oxidative activity Cells were seeded in serum-free and phenol-red-free cul- ture medium in 96-well plates for 24 hours, and then glu- cose was added to 30 mM. The cells were then washed in phenol-red-free culture medium containing 11 μM DCF- DA for 1 hour at 37oC. Well fluorescences were then meas- ured (480 nm excitation and 530 nm emission).
4. Measurement of GSH
GSH levels in cells were estimated by using 5-5'-dithio-
bis-(2-nitrobenzoic acid) (DTNB) reagent.17 Cells (6×105) were incubated in DMEM-serum for 24 hours. GECs were treated with EGCG (100 μM) 2 hours before adding glucose (30 mM). Cells were washed with PBS and 1 ml of 3%
perchloric acid was added and the cells were collected into microtubes. The tubes were placed on ice for 15 min and then centrifuged at 14,000×g (4oC, 10 min) and the super- natant was collected. To neutralize the supernatant (pH 7.5), the supernatant was transferred to fresh tubes con- taining 0.5 M KOH/9 mM Borax. To 250 μl of sample, 200 μl of DTNB reagent, 200 μl of NADPH, and 100 μl of GSSG reductase were added and the samples were incubated for 2-3 minutes in a 1-ml semimicro cuvette. The changes in absorbance at 412 nm were monitored for 3 min. The amount of GSH in the sample was calculated in nmole of GSH per mg protein from a standard curve obtained by us- ing known quantities of GSH.
5. Measurement of lipid peroxidation
The lipid peroxide level in cells was measured by using a thiobarbituric acid (TBA) assay, which monitors MDA production, according to the method of Buege and Aust.18 Cells (6×105) were incubated in DMEM-serum for 24 hours.
GECs were treated with EGCG (100 μM) 2 hours before adding glucose (30 mM). After 24 hours, the cells were washed and centrifuged at 14,000×g (4oC, 10 min), and 0.5 ml of the samples was added to 3 ml of 1% phosphoric acid and 1.0 ml of 0.6% TBA. These mixtures were heated in boil- ing water for 20 min. After cooling, the MDA was extracted by centrifugation at 15,000×g for 10 min and absorbance was measured at 532 nm. The results were expressed as nmole of MDA per mg protein.
6. Real-time reverse transcription (RT)-PCR
Total cellular RNA was isolated by using the single-step method and TRIzol reagent (Sigma, St. Louis, MO) and were reverse transcribed into cDNA. cDNA was then am- plified by using a PCR Thermal Cycler (Takara Bio Inc., Otsu, Shiga, Japan). The sequences of PCR primers used were as follows: rat TGF-β1 (sense, 5'-gac cgc aac aac gca atc tat gac-3'; antisense, 5'-gct gaa cca agg aga cgg aat aca- 3') and GAPDH (sense, 5'-gtc ttc acc acc atg gag aag g-3';
antisense, 5'- ctg gcc aag gtc atc cat ga-3'). PCR was con- ducted by using 30 amplification cycles of 95oC for 50 s, 60oC for 50 s, and 72oC for 50 s. After amplification, RT-PCR products were separated in 2% (w/v) agarose gels and stained with ethidium bromide. The sizes of the PCR prod- ucts for TGF-β1 and GAPDH genes were 303 bp and 198 bp, respectively. A PCR mixture containing SYBR-green (SYBR premix ExTaq, Takara Shuzo, Kyoto) was used.
PCR reactions in a final volume of 20 μl of reaction mixture contained 10 μl PCR mixture, 2 μl of specific primers (10 pM), 7 μl of nuclease-free water, and 1 μl of cDNA. Amplifi- cation was performed by using a Rotor-Gene 3000 (Corbett Research, Australia) programmed at 95oC for 15 min fol- lowed by 50 cycles of 95oC for 10 s, 60oC for 20 s, and 72oC for 30 s. Data from the reaction were collected and analyzed
EGCG on the Expression of TGF-β1, PKC α/βII and NF-κB in GECs
FIG. 1. Effects of EGCG on ROS production, content of GSH, and lipid peroxidation in glucose-treated GECs. (A) EGCG inhibited the ROS production by 30 mM glucose in GECs. Cells (1×104) were incubated in DMEM-serum-free media for 24 hours. Then the cells were treated with EGCG for 2 hours before adding glucose (30 mM). ROS pro- duction was assessed by using DCF-DA, as described in Materials and Methods. (B) EGCG inhibited the GSH suppression by 30 mM glucose in GECs. Cells (6×105) were treated with 30 mM glucose for 24 hours. GSH production was assessed by using DTNB reagent as described in Materials and Methods. (C) EGCG suppressed the LPO production by 30 mM glucose in GECs. LPO production was assessed by using TBA as described in Materials and Methods. *p＜0.0001 compared with the control group. †p＜0.0001, ‡p＜0.0005, com- pared with the 30 mM glucose-treated group. Values are expressed as means±S.E. of three independent experiments.
with Corbett Research Software (version 6.0). The com- parative critical threshold (Ct) values from quadruplicate measurements were used to calculate the gene expression, with normalization to GAPDH as an internal control.
Melting curve analysis was performed to enhance the spe- cificity of the amplification reaction.
7. Preparation of cell lysates and western blot analysis GECs treated with 30 mM glucose in the absence or pres- ence of EGCG were washed twice with ice-cold phos- phate-buffered saline solution and harvested by using a plastic scraper. Cells were lysed in lysis buffer on ice. The cell lysates obtained were then centrifuged at 13,200 rpm at 4oC, and the supernatants were transferred to fresh tubes and stored at −80oC until required. Protein concen- trations of lysates were determined by using BCA protein assay reagent. Proteins (10 μg) were resolved in 10% so- dium dodecyl sulfate-polyacrylamide gels at 120 V for 1 hour in a Mini-Protein II gel apparatus (Bio-Rad, Richmond, CA) and were then blotted onto a pre-wetted nitrocellulose membrane by using a Mini-Protein II gel apparatus. Mem- branes were incubated with washing buffer containing 5%
nonfat milk for at least 1 hour to block nonspecific protein binding. Primary mAb was diluted up to 1:1,000 in washing buffer and applied to membranes for 16 to 18 hours at 4oC.
After washing, blots were incubated with the appropriate
HRP-conjugated biotinylated secondary mAbs for 50 min at room temperature. Immunoreactive bands were vi- sualized by Western blotting by using Luminol Reagent (Santa Cruz Biotechnology).
8. Preparation of nuclear extracts and gel shift assay GECs were treated with EGCG followed by treatment with 30 mM glucose. Nuclear extracts were prepared from the cells as previously described, and extracts were frozen at −80oC. Protein concentrations in nuclear extracts were determined by use of BCA protein assay kits (Pierce Biotech., Rockford, IL). For gel shift assays, the nuclear extracts were incubated with biotin-labeled probes in binding buf- fer for 30 min at room temperature, according to the manu- facturer’s protocol (Panomics, Redwood City, CA). The probe sequences were as follows: NF-κB, AGTTGAGGGGACTT TCCCAGGC. For the competition control, we added excess unlabeled cold probes to the sample containing nuclear ex- tract and biotin-labeled probe. The samples were sepa- rated on a 6% polyacrylamide gel in 0.5% Tris-borate-EDTA buffer at 120 V for 60 min, transferred onto a nylon mem- brane, and fixed on the membrane by baking and UV cross- linking according to the manufacturer’s protocol (Panomics, Redwood City, CA). The biotin-labeled probe was detected with streptavidin-horseradish peroxidase.
FIG. 2. Effects of EGCG on the mRNA expression of TGF-β1. (A) GECs were pretreated with EGCG (100 μM), glucose (30 mM), or a combination of the two for 24 hours. Cellular RNAs were ex- tracted and the mRNA expression of TGF-β1 and GAPDH was ana- lyzed by RT-PCR. (B) The mRNA expression of TGF-β1 was meas- ured by real-time RT-PCR as described in Materials and Methods.
*p＜0.0001 compared with the control group. †p＜0.001, ‡p＜
0.0001, compared with the 30 mM glucose-treated group. Values are expressed as means±S.E. of three independent experiments.
FIG. 3. Effects of EGCG on the protein expression of TGF-β1. TGF- β1 and GAPDH expression was assessed by Western blotting with anti-TGF-β1 mAb or anti-GAPDH mAb, respectively. Results are shown in the top panels and quantified by densitometry (bottom panels). *p＜0.005 compared with the control group, †p＜0.001, ‡p
＜0.0005, compared with the 30 mM glucose-treated group. Values are expressed as means±S.E. of three independent experiments.
9. Statistics
Data are presented as the mean±S.E. Statistical signifi- cance was determined by using the ANOVA test (StatView;
Abacus Concepts Inc., Berkeley, CA). All experiments were performed at least three times on separate days and the da- ta presented herein are representative of the results ob- tained from all repetitions. p values of less than 0.05 were considered to be significant.
RESULTS
1. Effects of EGCG on high-glucose-induced ros produc- tion and content of GSH and LPO in GECs
To determine whether pretreatment with EGCG in- hibited high-glucose-induced ROS generation, we exam- ined cellular ROS levels in GECs. ROS production was sig- nificantly increased at a glucose concentration of 30 mM, whereas pretreatment with EGCG (100 μM) for 2 hours sig- nificantly decreased ROS production (Fig. 1A). Further- more, to determine whether EGCG pretreatment modu- lated the high-glucose-stimulated production of GSH and MDA, we measured GSH and LPO levels in GECs. As shown in Fig. 1B, treatment with 30 mM glucose reduced GSH contents in the GECs, which were increased by pre- treatment with EGCG (100 μM) for 2 hours. On the other
hand, LPO analysis revealed that production of MDA was reduced by pretreatment with EGCG (100 μM), but was in- creased by treating with 30 mM glucose alone (Fig. 1C). The production of MDA was not affected by treatment with 30 mM mannitol (an osmotic control) or 100 μM EGCG in 5 mM glucose.
2. Effects of EGCG on high-glucose-induced TGF-β1 ex- pression
TGF-β1 has been thought to mediate high-glucose-in- duced kidney damage. We investigated TGF-β1 mRNA and protein levels in GECs. We examined whether 30 mM glu- cose induced the mRNA expression of TGF-β1 in GECs by mRNA expression analysis performed by using real-time RT-PCR. TGF-β1 mRNA expression was increased by treatment with 30 mM glucose alone, whereas pretreat- ment with EGCG (10 and 100 μM) for 2 hours significantly reduced the expression of the TGF-β1 genes (Fig. 2). To fur- ther verify the effects of EGCG on the protein expression of TGF-β1, a Western blot analysis was performed. The pro- tein levels of TGF-β1 in the GECs were similar to the mRNA expression (Fig. 3).
3. Effects of EGCG on high-glucose-induced phosphory- lation of PKC α/βII
PKC α/βII has been suggested as being a downstream regulator of ROS under high-glucose conditions. Therefore, we investigated the relationship between PKC α/βII activa- tion, glucose-induced ROS generation, and TGF-β1 expre- ssion in GECs to determine whether EGCG plays a role in PKC α/βII phosphorylation. As shown in Fig. 4, marked in-
EGCG on the Expression of TGF-β1, PKC α/βII and NF-κB in GECs
FIG. 5. EGCG decreased the binding activities of NF-κB increased by 30 mM glucose in GECs. Cells (2×106) were incubated in DMEM- serum-free media for 24 hours. Then the cells were treated with EGCG for 2 hours before adding glucose (30 mM). Lane 1, 5 mM glucose medium nuclear extract; lane 2, 30 mM glucose-treated nuclear extract; lane 3, EGCG 100 μM alone treated nuclear ex- tract; lanes 4-6, EGCG 1-100 μM pretreated for 2 hours before treatment with 30 mM glucose nuclear extract; lane 7, 30 mM mannan-treated nuclear extract; lane 8, nuclear extract with com- peting unlabeled NF-κB probe.
FIG. 4. Effects of EGCG on the phosphorylation of PKC-α/βII. Cells (2×106) were incubated in DMEM-serum-free media for 24 hours.
Then the cells were treated with EGCG for 2 hours before adding glucose (30 mM). Phosphorylation of PKC-α/βII was assessed by Western blotting with anti-PKC-α mAb or anti-phospho-PKC-α/βII mAb, respectively. *p＜0.005 compared with the control group.
Results are shown in the top panels and quantified by densitometry (bottom panels). *p＜0.001 compared with the control group. †p
＜0.001 compared with the 30 mM glucose-treated group. Values are expressed as means±S.E. of three independent experiments.
creases in the phosphorylation of PKC α/βII were observed after treating GECs with 30 mM glucose for 24 hours.
Pretreatment with EGCG at 100 μM completely inhibited the 30 mM glucose-induced phosphorylation of PKC α/βII, which suggests that EGCG inhibits 30 mM glucose-in- duced alterations in the phosphorylation of PKC α/βII.
4. Effects of EGCG on high-glucose-induced alterations in the activation of NF-κB
Finally, we examined the effect of EGCG on the activa- tion of NF-κB. As shown in Fig. 5, treatment with 30 mM glucose resulted in activation of NF-κB in the GECs, which was prevented by pretreatment with EGCG (1, 10, 100 μM) in a dose-dependent manner. Complete blocking of NF-κB mobilization by the addition of the cold competitor in- dicated the specificity of NF-κB binding.
DISCUSSION
TGF-β1 signaling plays important roles in the pro- gression of kidney diseases and the accumulation of ex- tracellular matrix in diabetic nephropathy.19-21 In many re- nal cell types, high glucose stimulates the expression of TGF-β22 and increases the secretion of TGF-β1 that then acts on the cell in an autocrine manner to stimulate the ex- pression of extracellular matrix proteins.23 However, the mechanisms by which TGF-β1 operates in diabetic nephr- opathy are hard to define; signal transduction events seem
to be similar to the process induced by known pathways of stimulation of TGF-β1 synthesis (i.e., through smad, MAP kinases).24,25 We report here that the expression of mRNA and protein for TGF-β1 was significantly increased in GECs under the high-glucose condition when measured by real-time RT-PCR and Western blot analysis.
Recent studies suggest that phosphorylation of PKC con- tributes to up-regulation of the development of endothelial hyper-adhesiveness during acute hyperglycemia.26 Endo- thelial nitric oxide synthase is suppressed by activation of PKC-β in high-glucose conditions, resulting in a rapid re- duction in the production of nitric oxide in renal glomerular capillary endothelial cells.27 High glucose increases the ex- pression of PKC-β and PKC-δ in endothelial cells of bovine retina.28 To determine whether the effects of high glucose result from phosphoryration of PKC α/βII, we exposed cells to high-glucose conditions and performed Western blot analysis.
ROS may either act as second messengers for NF-κB acti- vation or directly induce their activation in a cell-specific manner.29 Recent studies have focused on the pro- or an- ti-apoptotic role of NF-κB that mediates the survival of cells. The transcription factor NF-κB plays a key role in reg- ulating apoptosis in numerous cell types by regulating the expression of a number of genes involved in proliferation and apoptosis. NF-κB activation is thought to play an im- portant role in the pathogenesis of renal failure. The pres- ent findings indicate that EGCG inhibited the NF-κB acti- vation induced by 30 mM glucose, which suggests that EGCG protects GECs from glucose-induced injury via down-regulation of NF-κB activation.
In summary, we demonstrated that increased production of ROS and MDA contents by high glucose in GECs was sup- pressed by the antioxidant EGCG. Moreover, GSH content, which was decreased by high glucose, was increased by EGCG. EGCG prevented TGF-β1 expression, phosphor- ylation of PKC α/βII, and NF-κB activation in GECs under high glucose concentrations. Our results provide evidence that EGCG could be a useful factor in modulating the injury to GECs by high glucose and that supplementation with an-
tioxidants is a promising complementary treatment that may exert beneficial effects in the diabetic kidney.
ACKNOWLEDGEMENTS
This work was supported by Chonnam Hospital Research Institute of Clinical Medicine (CUHRI-2008).
REFERENCES
1. Pavenstädt H, Kriz W, Kretzler M. Cell biology of the glomerular podocyte. Physiol Rev 2003;83:253-307.
2. Riser BL, Varani J, Cortes P, Yee J, Dame M, Sharba AK. Cyclic stretching of mesangial cells up-regulates intercellular adhesion molecule-1 and leukocyte adherence: a possible new mechanism for glomerulosclerosis. Am J Pathol 2001;158:11-7.
3. Jain S, De Petris L, Hoshi M, Akilesh S, Chatterjee R, Liapis H.
Expression profiles of podocytes exposed to high glucose reveal new insights into early diabetic glomerulopathy. Lab Invest 2011;91:488-98.
4. Noh H, Ha H. Reactive oxygen species and oxidative stress.
Contrib Nephrol 2011;170:102-12.
5. Stanton RC. Oxidative stress and diabetic kidney disease. Curr Diab Rep 2011;11:330-6.
6. Hawkins CL, Davies MJ. Generation and propagation of radical reactions on proteins. Biochim Biophys Acta 2001;1504:196-219.
7. Yu BP. Cellular defenses against damage from reactive oxygen species. Physiol Rev 1994;74:139-62.
8. Lan HY, Chung AC. Transforming growth factor-β and Smads.
Contrib Nephrol 2011;170:75-82.
9. Gopalakrishna R, Chen ZH, Gundimeda U. Modifications of cys- teine-rich regions in protein kinase C induced by oxidant tumor promoters and enzyme-specific inhibitors. Methods Enzymol 1995;252:132-46.
10. Barnes PJ, Karin M. Nuclear factor-kappaB: a pivotal tran- scription factor in chronic inflammatory diseases. N Engl J Med 1997;336:1066-71.
11. Yokozawa T, Nakagawa T, Oya T, Okubo T, Juneja LR. Green tea polyphenols and dietary fibre protect against kidney damage in rats with diabetic nephropathy. J Pharm Pharmacol 2005;57:
773-80.
12. Sutherland BA, Shaw OM, Clarkson AN, Jackson DN, Sammut IA, Appleton I. Neuroprotective effects of (-)-epigallocatechin gal- late following hypoxia-ischemia-induced brain damage: novel mechanisms of action. FASEB J 2005;19:258-60.
13. Yamabe N, Yokozawa T, Oya T, Kim M. Therapeutic potential of (-)-epigallocatechin 3-O-gallate on renal damage in diabetic nephropathy model rats. J Pharmacol Exp Ther 2006;319:228-36.
14. Fornoni A, Li H, Foschi A, Striker GE, Striker LJ. Hepatocyte growth factor, but not insulin-like growth factor I, protects podo- cytes against cyclosporin A-induced apoptosis. Am J Pathol
2001;158:275-80.
15. Kasinath BS, Maaba MR, Schwartz MM, Lewis EJ. Demonstra- tion and characterization of C3 receptors on rat glomerular epi- thelial cells. Kidney Int 1986;30:852-61.
16. Pugliese F, Singh AK, Kasinath BS, Kreisberg JI, Lewis EJ.
Glomerular epithelial cell, polyanion neutralization is associated with enhanced prostanoid production. Kidney Int 1987;32:57-61.
17. Ellman GL. Tissue sulfhydryl groups. Arch Biochem Biophys 1959;82:70-7.
18. Buege JA, Aust SD. Lactoperoxidase-catalyzed lipid peroxidation of microsomal and artificial membranes. Biochim Biophys Acta 1976;444:192-201.
19. Adhikary L, Chow F, Nikolic-Paterson DJ, Stambe C, Dowling J, Atkins RC, et al. Abnormal p38 mitogen-activated protein kinase signalling in human and experimental diabetic nephropathy.
Diabetologia 2004;47:1210-22.
20. Stambe C, Atkins RC, Tesch GH, Masaki T, Schreiner GF, Nikolic-Paterson DJ. The role of p38alpha mitogen-activated pro- tein kinase activation in renal fibrosis. J Am Soc Nephrol 2004;15:
370-9.
21. Hong SW, Isono M, Chen S, Iglesias-De La Cruz MC, Han DC, Ziyadeh FN. Increased glomerular and tubular expression of transforming growth factor-beta1, its type II receptor, and activa- tion of the Smad signaling pathway in the db/db mouse. Am J Pathol 2001;158:1653-63.
22. Sharma K, Ziyadeh FN. Hyperglycemia and diabetic kidney disease. The case for transforming growth factor-beta as a key mediator. Diabetes 1995;44:1139-46.
23. Wolf G, Sharma K, Chen Y, Ericksen M, Ziyadeh FN. High glu- cose-induced proliferation in mesangial cells is reversed by auto- crine TGF-beta. Kidney Int 1992;42:647-56.
24. Inoki K, Haneda M, Ishida T, Mori H, Maeda S, Koya D, et al. Role of mitogen-activated protein kinases as downstream effectors of transforming growth factor-beta in mesangial cells. Kidney Int Suppl 2000;77:S76-80.
25. Schiffer M, von Gersdorff G, Bitzer M, Susztak K, Böttinger EP.
Smad proteins and transforming growth factor-beta signaling.
Kidney Int Suppl 2000;77:S45-52.
26. Kouroedov A, Eto M, Joch H, Volpe M, Lüscher TF, Cosentino F.
Selective inhibition of protein kinase Cbeta2 prevents acute ef- fects of high glucose on vascular cell adhesion molecule-1 ex- pression in human endothelial cells. Circulation 2004;110:91-6.
27. Chu S, Bohlen HG. High concentration of glucose inhibits glomer- ular endothelial eNOS through a PKC mechanism. Am J Physiol Renal Physiol 2004;287:F384-92.
28. Park JY, Takahara N, Gabriele A, Chou E, Naruse K, Suzuma K, et al. Induction of endothelin-1 expression by glucose: an effect of protein kinase C activation. Diabetes 2000;49:1239-48.
29. Bowie A, O'Neill LA. Oxidative stress and nuclear factor-kappaB activation: a reassessment of the evidence in the light of recent discoveries. Biochem Pharmacol 2000;59:13-23.
